FACTORS AFFECTING THE INTRACELLULAR SYNTHESIS OF KYNURENINE.
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Near the time of pupation, autofluorescent kynurenine globules appear in the cells in the anterior region of the fatbody of Drosophila melanogaster. It has been reported previously that kynurenine synthesis may be induced in an additional group of fat cells by feeding the precursor tryptophan to Drosophila larvae, and that this induction of kynurenine production viewed within the fat cells is correlated with an increase in tryptophan pyrrolase activity. In the present report, conditions are outlined which result in the appearance of kynurenine in all of the fat cells. The number of cells in the fatbody which contain kynurenine is influenced by the quantity of tryptophan included in the diet, as well as by the developmental stage at the time of treatment and the duration of the feeding period on the inducer. Physical barriers modifying permeability, such as the membranous layer noted surrounding the fatbody, may be a factor in the regulation of the time and nature of the cellular induction of kynurenine synthesis. Another factor to be considered is the possibility of interference with the availability of tryptophan as a substrate or inducer for this synthesis within the cell. It is suggested that the occurrence of pteridines in some of the fat cells may modify the response of these cells to produce kynurenine, since pteridines as electron acceptors can complex with tryptophan as an electron donor. Kynurenine may be produced in the fat cells under in vitro conditions when they are incubated with L-tryptophan, but kynurenine is not formed when fat cells are incubated with D-tryptophan. The in vitro studies further demonstrate that induction of kynurenine synthesis may occur in fat cells isolated from young larvae in contrast, to in vivo conditions in which inducer does not effect an earlier appearance of kynurenine in the larval fatbody. (+info)
ALPHA SARCIN, A NEW ANTITUMOR AGENT. I. ISOLATION, PURIFICATION, CHEMICAL COMPOSITION, AND THE IDENTITY OF A NEW AMINO ACID.
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Isolation and purification procedures are given for the new antitumor agent, alpha sarcin. These procedures include the use of column ion exchange with a carboxylic resin (Amberlite IRC50), dialysis, decolorization with activated charcoal, gradient salt chromatography, salt removal, and drying from the frozen state. The final product has an activity of 800 sarcoma 180 mouse dilution units per mg. The amino acid composition of the purified material is reported. All of the usual amino acids found in proteins were present except methionine. In addition to the usual amino acids, an unknown amino acid was present in the acid hydrolysate. The latter was isolated, and was found to yield phenylalanine and kynurenine. This compound, which has been named "sarcinine," is extremely stable in 6 n hydrochloric acid in the absence of air, and is unstable in alkali. Sarcinine has also been found in two other antitumor peptides produced by aspergilli, and so may relate significantly to the antitumor properties of these peptides. (+info)
Comparative inhibition by substrate analogues 3-methoxy- and 3-hydroxydesaminokynurenine and an improved 3 step purification of recombinant human kynureninase.
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BACKGROUND: Kynureninase is a key enzyme on the kynurenine pathway of tryptophan metabolism. One of the end products of the pathway is the neurotoxin quinolinic acid which appears to be responsible for neuronal cell death in a number of important neurological diseases. This makes kynureninase a possible therapeutic target for diseases such as Huntington's, Alzheimer's and AIDS related dementia, and the development of potent inhibitors an important research aim. RESULTS: Two new kynurenine analogues, 3-hydroxydesaminokynurenine and 3-methoxydesaminokynurenine, were synthesised as inhibitors of kynureninase and tested on the tryptophan-induced bacterial enzyme from Pseudomonas fluorescens, the recombinant human enzyme and the rat hepatic enzyme. They were found to be mixed inhibitors of all three enzymes displaying both competitive and non competitive inhibition. The 3-hydroxy derivative gave low Ki values of 5, 40 and 100 nM respectively. An improved 3-step purification scheme for recombinant human kynureninase was also developed. CONCLUSION: For kynureninase from all three species the 2-amino group was found to be crucial for activity whilst the 3-hydroxyl group played a fundamental role in binding at the active site presumably via hydrogen bonding. The potency of the various inhibitors was found to be species specific. The 3-hydroxylated inhibitor had a greater affinity for the human enzyme, consistent with its specificity for 3-hydroxykynurenine as substrate, whilst the methoxylated version yielded no significant difference between bacterial and human kynureninase. The modified purification described is relatively quick, simple and cost effective. (+info)
Deficit of endogenous kynurenic acid in the frontal cortex of rats with a genetic form of absence epilepsy.
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The present studies sought to determine the concentrations of endogenous kynurenic acid (KYNA) and to measure the activity of kynurenine aminotransferases (KAT) I and II in the discrete brain regions of 3- and 6-month old WAG/Rij rats, a genetic model of absence epilepsy. Analogues experiments were performed using age-matched ACI rats, which served as a non-epileptic control. The age-dependent increase in KYNA concentration in the frontal cortex of WAG/Rij rats was considerably reduced in comparison to what was found in ACI rats. Consequently, the concentration of KYNA in the frontal cortex of epileptic rats was significantly lower than in non-epileptic controls. There were no such strain differences in other brain regions. The activities of KAT I and II also showed age-dependent increase with an exception for KAT II in the frontal cortex. Our data suggest that selective deficits of endogenous KYNA may account for increased excitability in the frontal cortex, which in turn may lead to the development of spontaneous spike-wave discharges in WAG/Rij rats. (+info)
Protein-bound kynurenine decreases with the progression of age-related nuclear cataract.
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PURPOSE: Posttranslational modification by UV filters is a key event in human lenses that appears to be largely responsible for normal age-dependent yellowing. It has been proposed that subsequent reactions of these covalently bound UV filters may also be involved in the genesis of age-related nuclear cataract. To examine this hypothesis, the levels of kynurenine-lysine and kynurenine-histidine were measured in both normal and cataractous human lenses. METHODS: Proteins isolated from the nuclei of normal lenses and lenses with and types I to IV nuclear cataract were hydrolyzed in 6 M HCl, and the levels of kynurenine-lysine and kynurenine-histidine were determined by HPLC. RESULTS: The content of kynurenine-lysine and kynurenine-histidine decreased substantially with the progression of age-related nuclear cataract. On average, levels of both kynurenine adducts were four times lower in advanced cataract (type IV) than in normal lenses. Simple autoxidation of the derivatives did not appear to be responsible for this decrease, because incubation in the presence of oxygen or H(2)O(2) did not affect adduct stability. CONCLUSIONS: Although protein-bound kynurenine accumulates over time in normal lenses, the levels attached to the proteins decrease significantly with the progression of age-related nuclear cataract. This finding suggests that in cataract there is a breakdown of the protein-bound adducts. Such further reactions of bound UV filters may contribute to the etiology of age-related nuclear cataract. (+info)
Double mechanism for apical tryptophan depletion in polarized human bronchial epithelium.
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Indoleamine 2,3-dioxygenase is an enzyme that catabolizes tryptophan to kynurenine. We investigated the consequences of IDO induction by IFN-gamma in polarized human bronchial epithelium. IDO mRNA expression was undetectable in resting conditions, but strongly induced by IFN-gamma. We determined the concentration of tryptophan and kynurenine in the extracellular medium, and we found that apical tryptophan concentration was lower than the basolateral in resting cells. IFN-gamma caused a decrease in tryptophan concentration on both sides of the epithelium. Kynurenine was absent in control conditions, but increased in the basolateral medium after IFN-gamma treatment. The asymmetric distribution of tryptophan and kynurenine suggested the presence of a transepithelial amino acid transport. Uptake experiments with radiolabeled amino acids demonstrated the presence of a Na(+)-dependent amino acid transporter with broad specificity that was responsible for the tryptophan/kynurenine transport. We confirmed these data by measuring the short-circuit currents elicited by direct application of tryptophan or kynurenine to the apical surface. The rate of amino acid transport was dependent on the transepithelial potential, and we established that in cystic fibrosis epithelia, in which the transepithelial potential is significantly more negative than in noncystic fibrosis epithelia, amino acid uptake was reduced. This work suggests that human airway epithelial cells maintain low apical tryptophan concentrations by two mechanisms, a removal through a Na(+)-dependent amino acid transporter and an IFN-gamma-inducible degradation by IDO. (+info)
Biochemical and phenotypic abnormalities in kynurenine aminotransferase II-deficient mice.
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Kynurenic acid (KYNA) can act as an endogenous modulator of excitatory neurotransmission and has been implicated in the pathogenesis of several neurological and psychiatric diseases. To evaluate its role in the brain, we disrupted the murine gene for kynurenine aminotransferase II (KAT II), the principal enzyme responsible for the synthesis of KYNA in the rat brain. mKat-2(-/-) mice showed no detectable KAT II mRNA or protein. Total brain KAT activity and KYNA levels were reduced during the first month but returned to normal levels thereafter. In contrast, liver KAT activity and KYNA levels in mKat-2(-/-) mice were decreased by >90% throughout life, though no hepatic abnormalities were observed histologically. KYNA-associated metabolites kynurenine, 3-hydroxykynurenine, and quinolinic acid were unchanged in the brain and liver of knockout mice. mKat-2(-/-) mice began to manifest hyperactivity and abnormal motor coordination at 2 weeks of age but were indistinguishable from wild type after 1 month of age. Golgi staining of cortical and striatal neurons revealed enlarged dendritic spines and a significant increase in spine density in 3-week-old mKat-2(-/-) mice but not in 2-month-old animals. Our results show that gene targeting of mKat-2 in mice leads to early and transitory decreases in brain KAT activity and KYNA levels with commensurate behavioral and neuropathological changes and suggest that compensatory changes or ontogenic expression of another isoform may account for the normalization of KYNA levels in the adult mKat-2(-/-) brain. (+info)
Human alpha-1-microglobulin is covalently bound to kynurenine-derived chromophores.
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Alpha-1-microglobulin carries a set of covalently linked chromophores that give it a peculiar yellow-brown color, fluorescence properties, and both charge and size heterogeneity. In this report it is shown that these features are due to the adducts with the tryptophan metabolite, 3-hydroxykynurenine, and its autoxidation products and that the modification is more pronounced in the protein isolated from urine of hemodialyzed patients. The light yellow amniotic fluid alpha-1-microglobulin acquires the optical properties and charge heterogeneity of the urinary counterpart following incubation with kynurenines. The colored amino acid adducts of urinary and amniotic fluid alpha-1-microglobulins were separated by chromatography after acid hydrolysis and analyzed by mass spectrometry. Human serum albumin samples, native and treated with 3-hydroxykynurenine in the presence of oxygen, were used as a control. The retention times and mass fragmentation products were compared, and a lysyl adduct with hydroxantommathin was identified in the urinary alpha-1-microglobulin and in the modified albumin samples. The more extensive modification of the urinary protein appears to be correlated with uremia, a condition in which the catabolism of tryptophan via the kynurenine pathway is increased, and the consequent rise in the concentration of its derivatives is accompanied by the oxidative processes due to the hemodialysis treatment. The oxidative derivatives of 3-hydroxykynurenine, which are known to act as protein cross-linking agents, are the likely cause of the propensity of urinary alpha-1-microglobulin to form dimers and oligomers. This process, as well as the redox properties of these metabolites, may contribute to the toxic effects of the kynurenine species. (+info)